**3. Conclusions**

Up to now, most of the HA-based carriers used as a VADS have been developed by making use of the "nonbioactive" properties, which has little physiological interference on the body and as such, is used just as constructive stuff. Moon's group formulated the HA-PEG-shelled cationic DOTAP/DOPE liposomes carrying F1-V, a candidate recombinant antigen for *Yersinia pestis*, as a stable and potent nasal VADS, which exhibited markedly decreased cytotoxicity associated with DOTAP liposomes to BMDCs, and when further incorporated with MPLA promoted BMDC maturation and induced a strong Th1/Th2-balanced immunoresponse toward Ags, as evidenced by high titers of F1-V-specific total IgG, IgG1, and IgG2c produced in intranasally immunized mice [69]. Huang's group engineered mLCP (the mannosylated lipid-calcium-phosphate NPs) and LPHa NPs (liposome-protamine-HA-anisamide NPs) for, respectively, targeting delivery of the tumor antigen Trp 2 peptide/CpG ODN to APCs and the TGF-β-silencing siRNA to tumor cells which overexpress sigma receptors with a ligand of anisamide [70]. They demonstrated that the delivery of Trp 2/CpG ODN to DCs by mLCPbased VADS elicited a potent systemic immune response to tumors in mice but generated, to later stage B16F10 melanoma, a marginal efficacy, which however, was remarkably boosted through silencing the immune-suppressive cytokine TGF-β in tumor cells with siRNA-loaded LPHa NPs to engender increased tumor infiltrating CD8+ T cells and decreased regulatory T cells within tumor microenvironment. Wu's group fabricated a microneedle array (MA) with HA with a deep cave formed in the basal portion of each microneedle, into which BCG (Bacille Calmette-Guerin bacillus) powder could be packaged directly, thus producing a painless VADS of MA-BCG, which after vaccination by patching on skin of mice caused no overt skin irritation, but elicited strong humoral and cellular immunity comparable to that of intradermal immunization [71]. Notably, other researchers showed in a clinical trial that HA-constructed MA containing trivalent influenza hemagglutinins (A/California/07/2009 (H1N1), A/Victoria/210/2009 (H3N2), and B/Brisbane/60/2008, 15 μg each) induced immune responses against A/H1N1 and A/H3N2 strains equal to that by subcutaneous injection groups without stirring severe local or systemic adverse reactions and engendered the efficacy against the B strain much stronger than that by the injection group, proving HA-MA a promising practical use as an easy and effective

Recently, Hahn's group conjugated an antigenic peptide of myostatin fragment (MstnF) to HA with a LMW (17 kDa) for transdermal vaccination against Duchenne muscular dystrophy (DMD), which is a neuromuscular disorder accompanied with muscle weakness and wasting with myostatin emerging as a key negative regulator [73]. In vivo experiments demonstrated that HA-MstnF conjugates efficiently penetrated into deep skin layers, and HA exerted a boosting effect on the immunization of MstnF in the transdermally vaccinated mice, which not only secreted high levels of antibody titers against myostatin but also showed a significant improvement in the pathological status of skeletal musculature as well as functional behaviors. Gonzalez-Aramundiz et al. prepared protamine/LMW HA (162 kDa) NPs using a mild ionic cross-linking technique and showed that in vitro Ag (rHBsAg)-loaded anionic NPs (protamine/HA of 1:4, w/w) induced the secretion of cytokines including TNFα, IL-1α, and IL-6 by macrophages more efficiently than the cationic NPs (protamine/HA of 4:1), whereas in mice, by either intramuscular or intranasal administration, the cationic NPs induced more robust immune responses than the anionic NPs did, as proved by the higher levels of the IgG

method to replace conventional injection systems [72].

38 Immunization - Vaccine Adjuvant Delivery System and Strategies

At present, various polymeric NP-based VADSs have been designed for delivering as well as adjuvanting vaccines to elicit robust Ag-specific humoral and cellular immunity at both systemic and mucosal levels to provide extensive protection against infectious pathogens. In particular, many types of polymeric NPs can be tailored as a multiple functional VADS to render Ags lysosome escape after APC uptake, allowing vaccine epitopes not only to avoid being degraded into null pieces but also to selectively bind to MHC-I or -II for presentation to dictate immune responses toward a Th1 and/or Th2 pathway to set up immunity fitting medical aims. Encouragingly, a few of the polymeric NP VADS-based subunit vaccines have been approved, as mile stones, for clinical vaccination, typical products including the virosomebased hepatitis A vaccine (Epaxal®) and influenza vaccine (Inflexal V®), VLP-based HBV vaccine and malaria vaccine. Undoubtedly, as many of the uncertainties and problematic issues associated with polymeric NPs, such as safety of synthetic materials, scale-up production, and cost of products, are ultimately resolved, more and more polymeric NP VADS-based vaccines will be developed and licensed to enter markets.
